Precise regulation of the initiation of DNA replication during S phase is essential to maintain genome integrity in eukaryotic cells. Therefore, understanding the mechanism and regulation of replication initiation is of critical importance for human health. In the last two decades, genetic approaches in yeast have identified specific DNA sequences that activate replication initiation, as well as several initiation factors (ORC, cdc6, MCM, cdc45) that are conserved in all eukaryotes. However, in many cases, the molecular functions of these factors as well as their regulation remain poorly understood. In addition, it is unlikely that all the factors required for replication initiation have been identified. To address these issues, a versatile cell-free system is essential. Using Xenopus laevis egg extracts, this investigator recently developed the first in vitro system that supports initiation of eukaryotic DNA replication in a completely soluble protein environment. This system supports -100 percent efficient DNA replication that is cell-cycle regulated. As such, it represents a unique opportunity to carry out a detailed biochemical analysis of replication initiation in eukaryotes. In Specific Aim 1, novel chromatin-binding assays will be used to develop a simple model for the spatial arrangement of initiation factors in the pre-replication complex. In Specific Aim 2, the mechanism by which cdk2/cyclin E protein kinase catalyzes association of cdc45 with origins of replication will be investigated. In Specific Aim 3, the time when DNA at the origin of replication is first denatured will be determined. In Specific Aim 4, the DNA helicase that unwinds the origin will be characterized. In Specific Aim 5, we examine the mechanism of a novel mode of chromatin binding by the MCM complex. Together, the proposed studies are expected to significantly advance understanding of eukaryotic replication initiation and provide a firm foundation to investigate how this process is regulated and how it might be mis-regulated during human disease.